Various methods and compositions for targeted modulation of gene expression of endogenous genomic DNA have been described. Targeted modulation of gene expression by DNA-binding proteins is described, for example, in U.S. Pat. Nos. 6,534,261; 6,607,882; 6,599,692; 6,689,558; 7,067,317; 7,947,873; 7,253,273; 7,358,085; 7,361,635; 7,534,775; 8,586,526 and U.S. Patent Publication No. 20110082093. Furthermore, targeted cleavage events using site-specific nucleases can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus. See, for example, U.S. Pat. Nos. 8,623,618; 8,034,598; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960 and U.S. application Ser. No. 14/278,903, the disclosures of which are incorporated by reference in their entireties for all purposes.
These methods often involve the use of engineered proteins that modulate expression of a target gene or engineered nuclease systems. In particular, the engineered transcription factors activate or repress targeted genes and the nucleases induce a double strand break (DSB) or a nick in a target DNA sequence such that repair of the break by non-homologous end joining (NHEJ) or homology directed repair (HDR) can result in the knock out of a gene and/or the insertion of a sequence of interest (targeted integration). Modulation and/or cleavage of endogenous genes can occur through the use of proteins and systems such as zinc finger protein transcription factors (ZFP-TFs), zinc finger nucleases (ZFNs), transcription-activator like effector transcription factors (TALE-TFs), CRISPR/Cas transcription factors (see, e.g., Perez-Pinera et al. (2013) Nature Methods 10:973-976), transcription-activator like effector nucleases (TALENs), Ttago nucleases or using the CRISPR/Cas system with an engineered crRNA/tracr RNA (‘single guide RNA’) to guide specific cleavage. Clinical trials using these engineered transcription factors containing zinc finger proteins have shown that these novel transcription factors are capable of treating various conditions (see, e.g., Yu et al. (2006) FASEB J. 20:479-481). Additionally, clinical trials using engineered zinc finger nucleases have also demonstrated therapeutic utility (see, e.g. Tebas et at (2014) New Eng J Med 370(10):901).
Gene modulation using these proteins and systems has the potential to treat various diseases and disorders, including, by way of example, HIV infection, cystic fibrosis, cancers such as glioblastomas, neuropathies, trinucleotide repeat disorders, HLA-related disorders, hemophilias, neurological conditions, pathogen infection, lysosomal storage diseases and hemoglobinopathies. See, e.g., U.S. Pat. No. 7,951,925; U.S. Patent Publication Nos. 20140017212; 20140093913; 20140080216; 20130145484, 20080188000, 20110082078, 20110082093, 20120196370, 20120128635, 20120214241, 20130253040. However, even in instances where the modulator preferentially binds (in a sequence-specific manner) to a target site mutant allele as compared to a wild-type allele (see, e.g., U.S. Patent Publication Nos. 20110082093 and 20130253040), over-expression of the modulator may result in binding to and/or undesired alteration of a wild-type sequence.
Thus, there remains a need for compositions and methods for regulating expression of exogenous gene-modulators within a cell to achieve optimal levels of expression of the modulators and subsequent modification of gene expression levels.